The present invention relates generally to methods for modifying the genome of animal cells, including human cells, and more particularly, to methods for modifying a genomic DNA sequence by homologous recombination using substantially isogenic DNA constructs.
Targeted gene disruption by homologous recombination has met with variable success in higher eukaryotes. While it has been possible to isolate cells which have stably incorporated exogenously prepared DNA sequences, in the vast majority of these cells, the DNA has integrated randomly into the genome rather than at the desired target site via homologous recombination. The ratio of the number of homologous recombinants to the total number of integration events varies, but typically, when there is no direct selection or enrichment for homologous recombinants, less than 1% of the integration events result from homologous recombination and ratios as low as 1 in 40,000 have been observed. Variations in the relative targeting efficiency have not been clearly attributable to differences in the length of homologous sequence present in the targeting constructs. Nor has any unequivocal correlation been documented between recombination efficiency and transcriptional activity of the target gene or chromosomal location of the target gene.
If the homologous recombinants can only be obtained amidst a large background of random integration events, then : it may be impractical, if not impossible, to effectively target many genomic sequences. The approaches taken to overcoming this problem have focused on developing special strategies to screen or select homologous recombinants from the large background of non-homologous or random integration events. In a few situations in which the targeted gene is itself a dominant selectable marker, it may be feasible to select directly for homologous recombinants. For example, knocking out the hprt gene (encoding hypoxanthine phosphoribosyl transferase) results in increased tolerance of the base analog 6-thioguanine (Thomas, K. and M. Capecchi, Cell 51:503-512 (1987). However, such particularized methods are not widely applicable. Other selection procedures aim at the enrichment for the desired homologous recombination event by suppressing colony formation due to random integrations of the targeting construct. In single selection protocols, the targeting constructs contain a marker gene, typically conferring drug resistance, deprived of transcriptional and/or translational start signals, in ""such a way that the juxtaposition of the marker gene and functional expression signals would be obtained on homologous recombination but only rarely on random integration. Sedivy, J., and P. Sharp, Proc. Nat""l Acad. Sci. USA 86:227-231 (1989). The double or xe2x80x9cpositive/negativexe2x80x9d selection procedure developed by Capecchi and co-workers makes use of an autonomously expressed marker gene, but the targeting construct is flanked by a second gene which is detrimental to the cell and which tends to be lost on homologous recombination but not on random integration. Mansour, S., at al., Nature 336:348-352 (1988).
Another approach has involved the use of screening procedures based on the polymerase chain reaction (xe2x80x9cPCRxe2x80x9d), in which pools of cells are tested for potential homologous recombinants using pairs of primers which will be juxtaposed only if homologous recombination has occurred. Any pools containing potential homologous recombinants are then sub-divided and the procedure is continued until a small enough pool of cells can be analyzed individually. Zimmer, A., et al., Nature 338:150-153 (1989); and Joyner, A., et al., Nature 338:153-156 (1989). Besides the labor involved in screening, the PCR protocols also require that appropriate regions of the DNAs have been sequenced and that oligonucleotide primers be obtained.
The relative inefficiency of homologous recombination is even more problematic when working with cells that are not easily reproduced in vitro and for which the aforementioned selection and screening protocols may be impractical, if not impossible. For example, there are a large variety of cell types, including many stem cell types, which are difficult or impossible to clonally reproduce in vitro. If the relative frequency of homologous recombination itself could be improved, then it might be feasible to target a variety of cells which are not amenable to specialized isolation techniques such as positive/negative selection or PCR screening. (See, W091/01140, which is incorporated herein by reference.)
Thus, there remains a significant need for gene targeting systems in which homologous recombinants can be routinely and efficiently obtained at a high enough frequency to obviate the necessity of special selection and screening protocols. The present invention fulfills these and other needs.
The present invention provides novel methods for modifying the genome of an animal cell comprising the steps of: constructing a DNA molecule in which desired sequence modifications are contained in a segment of DNA (a xe2x80x9ctargeting DNAxe2x80x9d) that is substantially isogenic with a DNA in the cell genome (a xe2x80x9ctarget DNAxe2x80x9d); introducing the targeting DNA construct into the cell (e.g., by microinjection, electroporation, transfection, or calcium phosphate precipitation); and selecting cells in which the desired sequence modifications have been introduced into the genome via homologous recombination.
Preferably, the targeting DNA will be derived from a cell line that is closely related to the cell line which is being targeted; so that the sequence of the targeting DNA is substantially identical with the sequence of the target DNA (except for the desired sequence modifications). By using substantially isogenic targeting DNA, a substantial fraction of the cells in which integration has occurred will have undergone homologous recombination between the targeting DNA sequence and the target DNA sequence. Since the integration events are thereby enriched for homologous recombinates, it is possible to forego the use of special selection and screening protocols used to isolate rare homologous recombinants from a large background of non-homologous integration events.
Although the present invention has been applied to laboratory mice strains such as BALB/c and 129, the invention will be even more useful for gene targeting in non-murine animals. The typical mouse strains used in laboratories tend to be fairly inbred and, as a result, there is smaller likelihood of sequence divergence in an allele derived from different lines (see, e.g., Bishop, C., et al., Nature 315:70-72 (1985)). In contrast, many other animals are not so inbred, and there is a greater chance of sequence divergence between alleles derived from different individuals. The restriction fragment length polymorphisms (xe2x80x9cRFLPsxe2x80x9d), useful in xe2x80x9cfingerprintingxe2x80x9d human DNA, are an example of this phenomenon in a non-inbred species.
A preferred cell type for targeting the genome of a mammalian organism is the embryonic stem cell. Preferably, the DNA construct contains an antibiotic resistance marker and the cells are first selected on a medium containing the antibiotic.
The present invention also provides novel methods for creating genetically modified animals comprising the steps of: modifying the genome of embryonic stem cells derived from the animal, as described above; introducing the modified embryonic stem cells into blastocysts derived from the same species of animal; and using a pseudo-pregnant female to carry the chimeric animal to term. The resulting chimeric animal can in turn be bred to obtain non-chimeric animals in which the desired genetic alteration has -been stably inherited through germ-line transmission.
The present invention can also be used for the direct targeting of animal zygotes. The targeting DNA can be introduced by, for example, microinjection, and then, with mammals for example, the modified zygotes can be transferred to pseudo-pregnant females capable of carrying the animal to term. Similarly, for somatic gene therapy, the genome of somatic cells of an animal is directly modified using the substantially isogenic targeting DNA and then the modified cells are introduced into the same or a different animal.
In another aspect, the present invention provides cells exhibiting a recombination event at a preselected native target DNA site in the cell genome. Thus, in view of the increased efficiency of recombination utilizing the methods of the present invention, a collection of cells having undergone a recombination event will exhibit between about 10-90%, typically at least about 30 to 50%, recombination. The cells exhibiting the desired characteristics may be selected for and isolated in accordance with standard techniques, and grown into animals.